Method to diagnose and treat pathological conditions resulting from deficient ion transport such as pseudohypoaldosteronism type-1

ABSTRACT

The present invention is based, in part, on the identification of the roles of the human ATP-sensitive K +  channel, ENaC in causing pathological condition associated with abnormal ion transport, particularly PHA1. The present invention specifically provides the amino acid sequence of several human altered variants of the ENaC protein as well as the nucleotide sequence that encodes these variants that can be used in diagnosing ion transport disorders.

This application is a 371 filing of PCT/US98/04681 filed Mar. 11, 1998.This application is related to U.S. Application Ser. No. 60/040,171,filed on Mar. 11, 1997, which is herein incorporated by reference in itsentirety.

TECHNICAL FIELD

The invention relates to the fields of detecting and treating homozygousand heterozygous genetic deficiencies in ion transport, particularlyalterations in nucleic acid molecules and proteins that give rise tovarious forms of pseudohypoaldosteronism type-1 (PHA1). Morespecifically, the invention provides compositions and methods fordetermining whether an individual is affected by or carriers a mutationa gene that encodes a protein involved in ion transport.

BACKGROUND ART

Background

Pseudohypoaldosteronism type I (PHA1) is a rare salt wasting diseasecharacterized by an often fulminate presentation in the neonatal periodwith dehydration, hyponatremia, hyperkalaemia, metabolic acidosis,failure to thrive and weight loss despite normal renal glomerularfiltration and adrenal function (Cheek, D. et al. Archives of Dis. inChildhood 33:252-256 (1958); Dillon, M. J. et al. Archives of Dis. inChildhood 55:427-434 (1980); Popow, C., et al. Acta Paediatr. Scand.77:136-141 (1988); Speiser, P. W., Stoner, E. & New, M. I.Pseudohypoaldosteronism: a review and report of two new cases. In:Mechanisms and clinical aspects of steroid hormone resistance. (eds.Chrousos, G. P., Loriaux, D. T. & Lipsett, M. B.) 173-195. (PlenumPress, New York), 1986). PHA1 is suspected when these infants fail torespond to mineralocorticoids, and the diagnosis is supported by thefinding of an elevated plasma aldosterone concentration and plasma reninactivity (Dillon, M. J. et al. Archives of Dis. in Childhood 55:427-434(1980); Popow, C., et al. Acta Paediatr. Scand. 77:136-141 (1988);Speiser, P. W., Stoner, E. & New, M. I. Pseudohypoaldosteronism: areview and report of two new cases. In: Mechanisms and clinical aspectsof steroid hormone resistance. (eds. Chrousos, G. P., Loriaux, D. T. &Lipsett, M. B.) 173-195. (Plenum Press, New York), 1986). Treatmentincludes sodium chloride supplementation and treatment with anion-binding resin or dialysis to reduce life-threatening hyperkalaemia(Cheek, D. et al. Archives of Dis. in Childhood 33:252-256 (1958);Dillon, M. J. et al. Archives of Dis. in Childhood 55:427-434 (1980);Popow, C., et al. Acta Paediatr. Scand. 77:136-141 (1988); Speiser, P.W., Stoner, E. & New, M. I. Pseudohypoaldosteronism: a review and reportof two new cases. In: Mechanisms and clinical aspects of steroid hormoneresistance. (eds. Chrousos, G. P., Loriaux, D. T. & Lipsett, M. B.)173-195. (Plenum Press, New York), 1986; Donnell, G. N., et al. Am. J.Dis. Child. 97:813-828 (1959); Mathew, P. M., et al. ClinicalPediatrics. 1:58-60 (1993)). Death in the neonatal period is common ifthe diagnosis is not made.

PHA1 kindreds showing both autosomal recessive and dominant transmissionhave been described (Hanukoglu, A. J. Clin. Endocrin. & Metab.73,936-944 (1991)). Cases in recessive kindreds typically showmineralocorticoid resistance in the kidney, sweat and salivary glands,and colonic mucosa (Speiser, P. W., Stoner, E. & New, M. I.Pseudohypoaldosteronism: a review and report of two new cases. In:Mechanisms and clinical aspects of steroid hormone resistance. (eds.Chrousos, G. P., Loriaux, D. T. & Lipsett, M. B.) 173-195. (PlenumPress, New York), 1986; Hanukoglu, A. J. Clin. Endocrin. & Metab.73,936-944 (1991); Hanukoglu, A., et al. J. Pediatr. 125: 752-755(1994); Hogg, R. J., et al. Pediatric Nephrology 5:205-210 (1991));where measured, parents of these cases have had normal aldosterone andrenin levels (Speiser, P. W., Stoner, E. & New, M. I.Pseudohypoaldosteronism: a review and report of two new cases. In:Mechanisms and clinical aspects of steroid hormone resistance. (eds.Chrousos, G. P., Loriaux, D. T. & Lipsett, M. B.) 173-195. (PlenumPress, New York), 1986; Hanukoglu, A. J. Clin. Endocrin. & Metab.73,936-944 (1991)). In contrast, kindreds supporting dominanttransmission have also been reported, and in some of these have beenshown to have disease limited to the kidney (Speiser, P. W., Stoner, E.& New, M. I. Pseudohypoaldosteronism: a review and report of two newcases. In: Mechanisms and clinical aspects of steroid hormoneresistance. (eds. Chrousos, G. P., Loriaux, D. T. & Lipsett, M. B.)173-195. (Plenum Press, New York), 1986; Hanukoglu, A. J. Clin.Endocrin. & Metab. 73,936-944 (1991); Limal, J. M., et al. Lancet 1:51(1978); Hanukoglu, A., et al. Lancet 1:1359 (1978)). Clinical signs andmetabolic abnormalities of some patients improve in the first severalyears of life, allowing discontinuation of therapy (Cheek, D. et al.Archives of Dis. in Childhood 33:252-256 (1958); Dillon, M. J. et al.Archives of Dis. in Childhood 55:427-434 (1980); Speiser, P. W., Stoner,E. & New, M. I. Pseudohypoaldosteronism: a review and report of two newcases. In: Mechanisms and clinical aspects of steroid hormoneresistance. (eds. Chrousos, G. P., Loriaux, D. T. & Lipsett, M. B.)173-195. (Plenum Press, New York), 1986; Donnell, G. N., et al. Am. J.Dis. Child. 97:813-828 (1959); Hanukoglu, A. J. Clin. Endocrin. & Metab.73,936-944 (1991)); it has been suggested that these patients are mostoften those with dominant transmission (Hanukoglu, A. J. Clin. Endocrin.& Metab. 73,936-944 (1991)).

The pathogenesis of this syndrome has not been elucidated. The triad ofrenal salt wasting, hyperkalaemia and failure to respond tomineralocorticoids is most compatible with a renal defect in the distalnephron (Cheek, D. et al. Archives of Dis. in Childhood 33:252-256(1958); Rösler, A. J. Clin. Endocrin. & Metab. 59:689-700 (1984)). Whilemineralocorticoid receptor levels in affected patients have been foundto be low (Armanini, D. et al. N. Eng. J Med. 313:1178-1181 (1985);Kuhnle U. et al. J. Clin. Endocrin. & Metab. 70:638-641 (1990); Bosson,D. et al. Acta Endo. 113:S376-S381 (1986)) molecular studies haverevealed no evidence for a primary defect in the mineralocorticoidreceptor (Komesaroff, P. A., et al. J. Clin. Endocrin. & Metab. 79:27-31(1994); Zennaro, M. C., et al. J. Clin. Endocrin. & Metab. 79:32-38(1994)).

Electrogenic transepithelial sodium transport is the rate limiting stepin sodium reabsorption in the distal nephron, the distal colon, salivaryand sweat glands, and lung epithelia (Horisberger, J. D., et al. CellPhysiol. Biochem. 32:283-294 (1993)). In the kidney, this electrogenicsodium transport is positively regulated by aldosterone (Rossier, B. C.& Palmer, L. G. Mechanism of aldosterone action on sodium and potassiumtransport. In: The Kidney, physiology and pathophysiology (eds. Seldin,D. W. and Giebisch, G.) 1373-1409 (Raven Press, New York, 1992)) and ismediated by the amiloride-sensitive epithelial sodium channel (ENaC).This channel composed of at least three subunits of similar structure(Canessa, C. M., et al. Nature 361:467-470 (1993); Canessa, C. M. et al.Nature 367:463-467 (1994)), each with intracellular amino and carboxytermini, two transmembrane spanning domains, and a large extracellularloop. In humans, αENaC is present on human chromosome 12, while b and gare tightly linked on chromosome 1622.

Mutations resulting in constitutive activation of ENaC activity havebeen shown to cause an autosomal dominant form of hypertension, Liddle'ssyndrome (Shimkets, R. A. et al. Cell 79:407-414 (1994); Hansson, J. H.et al. Nature Genetics 11:76-82 (1995); Hansson, J. H. et al. Proc. Nat.Acad. Sci. USA 92:11495-11499 (1995); Schild, L. et al. Proc. Natn.Acad. Sci. USA 92:5699-5703 (1995)), which is characterized by volumeexpansion, hypokalaemia and alkalosis.

The present invention provides compositions and methods that can be usedto differentiate and diagnose ion transport deficiencies, particularlyPHA1. The present invention further provides methods and compositionsthat can be used to identify heterozygous carriers for this disorder.Carriers, though not displaying severe clinical symptoms, nonethelessdisplay mild to moderate pathologies.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the identification of therole of the epithelial sodium channel (ENaC) in pathological conditionassociated with abnormal ion transport, particularly PHA1, hypokalaemicalkalosis, hypokalaemic acidosis and salt wasting. The present inventionspecifically provides the amino acid sequences of several humanwild-type and altered variants of the ENaC proteins as well as thenucleotide sequence that encodes these variants. These proteins andnucleic acid molecules can be used in diagnosing ion transport disordersand in developing methods and agents for treating these pathologies.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1. PHA1 Kindreds

The family relationships of 5 PHA1 kindreds in which mutations have beenidentified are shown. All affected subjects are the product ofconsanguineous union. Subjects classified as affected are indicated byfilled symbols; unaffected subjects are indicated by unfilled symbols;deceased subjects are indicated by a diagonal line; index cases areindicated by an arrow; living subjects who were not sampled areindicated by dots. Within each kindred, each sampled individual isidentified by a unique number, which is shown above and to the left oftheir respective symbol. Below each symbol, the SSCP genotype at eitherαENaC (PHA K10, K13, K14 and K3) or βENaC (PHA K8) is shown. Thesymbol + denotes the normal SSCP variant, and the numbers 1, 2, and 3indicate the αENaC codon 68 frameshift, αENaC codon 508 stop, and βENaCG37S mutations, respectively.

FIG. 2. Mutations in ENaC Subunits in PHA1 Patients

In each panel, variants identified by SSCP in PHA1 kindreds are shown.Individuals are numbered as in FIG. 1, and representative autoradiogramsare shown. SSCP genotypes, as well as marker genotypes, were confirmedfrom at least two independent amplifications for each individual.Affected individuals are indicated by an asterisk, and the novel SSCPvariants that are homozygous in affected subjects are indicated byarrows; these variants are numbered as in FIG. 1. At the bottom of eachpanel, the DNA sequence of the mutant allele (left) and correspondingwild-type allele (right) is shown; deleted bases are indicated by abracket in panel A, and single base substitutions are indicated byasterisks in panels B and C SEQ ID NO: 1 (CCACCATCCACGG); SEQ ID NO: 2(CCACCACACGG); SEQ ID NO: 3 (CTGTCGCGA); SEQ ID NO: 4 (CTGTCACGA); SEQID NO: 5 (CACGGCCCC); and SEQ ID NO: 6 (CACAGCCCC).

2 a. Affected subjects in PHA kindreds K10, K13 and K14 are homozygousfor the same 2 base pair deletion introducing a frameshift at codon 68of αENaC, and this variant cosegregates with the disease. The DNAsequence extending from the last two bases of codon 66 to the first twobases of codon 70 of the wild type sequence are shown in the senseorientation. The last two bases of codon I68 are absent in the mutant.

2 b. Homozygous variant in αENaC introducing a premature terminationcodon at codon 508 of PHA K3. DNA sequence encoding amino acids 507-509are shown in the antisense orientation; in the sense orientation, CGAencoding R508 is mutated to TGA, encoding stop 508.

2 c. Homozygous variant in βENaC encoding G37S in affected subjects ofPHA K8. DNA sequence of codons 36-38 are shown in the sense orientation.The sequence GGC encoding G37 is mutated to AGC encoding S37.

FIG. 3. Consequences of Mutations Identified in PHA1 Kindreds

3 a. The effects of mutations in α and βENaC in PHA1 kindreds are shown.ENaC subunits are drawn as spanning the plasma membrane twice (CanessaC. M., et al. Am. J. Ped. 267:C1682-169 (1994)), and amino and carboxyltermini are indicated. Arrows indicate the position of identifiedmutations in each subunit. The kindreds in which each mutation is foundare indicated. Mutation in αENaC introduces a frameshift mutation atcodon 68 proximal to the first transmembrane domain; this mutation isfound in 3 kindreds. A mutation in PHA K3 introduces a prematuretermination codon into the extracellular domain of αENaC, and a mutationin βENaC in PHA K8 introduces a missense mutation, changing glycine atresidue 37 to serine.

3 b. G37S mutation in βENaC occurs in a conserved ENaC segment. Aminoacid sequences preceding the first transmembrane domain of differentmembers of the ENaC family are shown. The prefix h, r, and x denotegenes from human, rat and Xenopus laevis, respectively (Canessa, C. M.,et al. Nature 361:467-470 (1993); Canessa, C. M. et al. Nature367:463-467 (1994); McDonald, F. J., et al. Am. J Physiol. 268:L728-734(1994); McDonald, F. J., et al. Am. J Physiol. 268: C1157-C1163 (1995);Puoti, A. et al. Am J Physiol. 269:C188-C197 (1995); Waldmann, R., etal. J. of Biol. Chem. 270:27411-27414 (1995)); mec-10 and Deg-1 are fromC. elegans (Huang, M. et al. Nature 367:467-470 (1994); Chalfie, M. etal. Nature 345:410-416 (1990)). Those residues that are identical in a,b and g subunits from all species are shaded. The completely conservedglycine at position 37 of hbENAC is mutated to serine in PHA kindred 8.SEQ ID NO: 7 (hβENaC); SEQ ID NO: 8 (rβENaC); SEQ ID NO: 9 (xβENaC); SEQID NO: 10 (hαENaC); SEQ ID NO: 11 (rαENaC); SEQ ID NO: 12 (xαENaC); SEQID NO: 13 (hγENaC); SEQ ID NO: 14 (rγENaC); SEQ ID NO: 15 (xγENaC); SEQID NO: 16 (hδENaC); SEQ ID NO: 17 (mec-10); and SEQ ID NO: 18 (Deg-1).

FIG. 4. Effect of βENaC G37S on Amiloride-sensitive Na⁺Channel Activityin Xenopus Oocytes

cRNAs encoding normal or mutant ENaC subunits were coinjected intoXenopus oocytes, and the resulting amiloride-sensitive sodium currentwas measured. βENaC containing the G37S mutation was coexpressed with αand γ subunits (represented as αβ37Sγ). Oocytes injected either withnormal α, β and γ or only α and γ subunits (αβγ and αγ, respectively)served as controls. The mean of the absolute values of theamiloride-sensitive sodium current obtained from 33 to 35 oocytes from 5different batches of oocytes is shown. Error bars represent the SEM. Thep values for t-tests comparing activity of mutant and wild-type channelsare indicated.

MODES OF CARRYING OUT THE INVENTION

I. General Description

The present invention is based, in part, on the identification of therole of the epithelial sodium channel (ENaC) in pathological conditionsassociated with abnormal ion transport, particularly PHA1, hypokalaemicacidosis and salt wasting. The present invention specifically providesthe amino acid sequences of several human wild-type and altered variantsof the α, β and γ subunits of the ENaC protein, as well as thenucleotide sequence that encodes these variants. These proteins andnucleic acid molecules can be used in diagnosing ion transport disordersand in developing methods and agents for treating these pathologies.

II. Specific Embodiments

A. ENaC Protein

Prior to the present invention the art had identified: the amino acidsequence of the three subunits of human ENaC. However, prior to thepresent invention, (1) no one had identified that alterations in humanvariants of the ENaC protein that produce inactive ENaC result in viableindividuals that suffer from pathologies caused by abnormal iontransport; (2) no one had characterized naturally occurring humanwild-type variants of the subunits of ENaC; (3) no one had characterizedhuman altered variants of the ENaC protein that yielded inactive ENaC;and (4) no one had shown that pathological conditions that are a resultof abnormal ion transport, such as PHA1, could be identified byanalyzing a sample for the presence of a wild-type or an altered variantof the ENaC protein. The present invention provides, in part, the aminoacid sequences of wild-type human ENaC protein and altered variants ofthe human ENaC protein that give rise to ion transport deficiencies, aswell as the nucleotide sequence of the encoding nucleic acid molecules.

In one embodiment, the present invention provides the ability to producepreviously unknown altered variants of the human ENaC proteins using thecloned nucleic acid molecules herein described.

As used herein, a wild-type human ENaC protein refers to a protein thathas the amino acid sequence of a wild-type allelic variant of humanENaC. The ENaC protein is comprised of 3 subunits: the α, β and γsubunits, referred to herein as αENaC, βENaC and γENaC, respectively.For the sake of convenience, the collective subunits will be referred toas the ENaC protein. The wild-type ENaC proteins of the presentinvention include the specifically identified and characterized variantherein described as well as allelic variants that can be isolated andcharacterized without undue experimentation following the methodsoutlined below. For the sake of convenience, all of the wild-type humanENaC proteins of the present invention will be collectively referred toas the wild-type ENaC proteins or the wild-type human ENaC proteins ofthe present invention.

The term “wild-type human ENaC proteins” includes all naturallyoccurring allelic variants of the human ENaC protein that posses normalENaC activity. Allelic variants, though possessing a slightly differentamino acid sequence than those recited above, will posses the ability tobe a sodium transporter. Typically, allelic variants of the wild-typeENaC protein will contain conservative amino acid substitutions from thewild-type sequences herein described or will contain a substitution ofan amino acid from a corresponding position in an ENaC homologue (anENaC protein isolated from an organism other than human).

As used herein, a mutated or altered human ENaC protein refers to aprotein that has the amino acid sequence of a mutated or altered allelicvariant of human ENaC that produces an ENaC protein with reduced iontransport capability. FIG. 3 provides the amino acid sequences ofseveral mutated or altered allelic variants of each of the threesubunits of human ENaC protein. The mutated or altered ENaC proteins ofthe present invention include those specifically identified andcharacterized herein as well as allelic variants that can be isolatedand characterized without undue experimentation following the methodsoutlined below. For the sake of convenience, all of the mutated oraltered human ENaC proteins of the present invention will becollectively referred to as mutated or altered human ENaC proteins orthe mutated or altered human ENaC proteins of the present invention.

The term “mutated or altered human ENaC proteins” includes all naturallyoccurring allelic variants of the human ENaC protein that do not possesnormal ENaC activity. Mutated or altered allelic variants will be not beable to transport sodium or will transport sodium at a reduced rate whencompared to wild-type ENaC. Typically, mutated or altered ENaC proteinwill contain: non-conservative amino acid substitutions from thewild-type sequences herein described; a substitution of an amino acidother than the amino acid found in a corresponding position in an ENaChomologue (an ENaC protein isolated from an organism other than human);a frame shift mutation, an insertion of a stop codon; or a deletion orinsertion of one or more amino acids into the ENaC sequence.

The ENaC proteins of the present invention (wild-type and mutatedvariants) are preferably in isolated from. As used herein, a protein issaid to be isolated when physical, mechanical or chemical methods areemployed to remove the ENaC protein from cellular constituents that arenormally associated with the protein. A skilled artisan can readilyemploy standard purification methods to obtain an isolated ENaC protein.The nature and degree of isolation will depend on the intended use.

The cloning of ENaC encoding nucleic acid molecules makes it possible togenerate defined fragments of the ENaC proteins of the presentinvention. As discussed below, fragments of the ENaC proteins of thepresent invention are particularly useful in generating domain specificantibodies, in identifying agents that bind to an ENaC protein and inidentifying ENaC intra- or extracellular binding partners.

Fragments of the ENaC proteins can be generated using standard peptidesynthesis technology and the amino acid sequences disclosed herein.Alternatively, recombinant methods can be used to generate nucleic acidmolecules that encode a fragment of the ENaC protein. by FIG. 3identifies amino acid residues that are altered from wild-type residuesin altered variants of the ENaC proteins described herein. Fragmentscontaining these residues/alterations are particularly useful ingenerating altered variant specific anti-ENaC antibodies.

As described below, members of the ENaC family of proteins can be usedfor, but are not limited to: 1) a target to identify agents that blockor stimulate ENaC activity, 2) a target or bait to identify and isolatebinding partners that bind an ENaC protein, 3) identifying agents thatblock or stimulate the activity of an ENaC protein and 4) an assaytarget to identify ENaC mediated activity or disease.

B. Anti-ENaC Antibodies

The present invention further provides antibodies that selectively bindone or more of the ENaC proteins of the present invention. The mostpreferred antibodies will bind to an altered variant of an ENaC proteinbut not to a wild-type variant or will bind to a wild-type variant of anENaC protein but not to an altered variant. Anti-ENaC antibodies thatare particularly contemplated include monoclonal and polyclonalantibodies as well as fragments containing the antigen binding domainand/or one or more complement determining regions.

Antibodies are generally prepared by immunizing a suitable mammalianhost using an ENaC protein, or fragment, in isolated or immunoconjugatedvariant (Harlow Antibodies, Cold Spring Harbor Press, NY (1989)). FIG. 3identifies several regions of the ENaC protein that have been shown tobe mutated in various altered variants of the ENaC protein describedherein. Fragments containing these residues are particularly suited ingenerating wild-type or mutated-variant specific anti-ENaC antibodies.

Methods for preparing a protein for use as an immunogen and forpreparing immunogenic conjugates of a protein with a carrier such asBSA, KLH, or other carrier proteins are well known in the art. In somecircumstances, direct conjugation using, for example, carbodiimidereagents may be used; in other instances linking reagents such as thosesupplied by Pierce Chemical Co., Rockford, Ill., may be effective.

Administration of the ENaC immunogen is conducted generally by injectionover a suitable time period and with use of a suitable adjuvant, as isgenerally understood in the art. During the immunization schedule,titers of antibodies can be taken to determine adequacy of antibodyformation.

While the polyclonal antisera produced in this way may be satisfactoryfor some applications, for pharmaceutical compositions, monoclonalantibody preparations are preferred. Immortalized cell lines whichsecrete a desired monoclonal antibody may be prepared using the standardmethod of Kohler and Milstein or modifications which effectimmortalization of lymphocytes or spleen cells, as is generally known.The immortalized cell lines secreting the desired antibodies arescreened by immunoassay in which the antigen is the ENaC protein orpeptide fragment. When the appropriate immortalized cell culturesecreting the desired antibody is identified, the cells can be culturedeither in vitro or by production in ascites fluid.

The desired monoclonal antibodies are then recovered from the culturesupernatant or from the ascites supernatant. Fragments of themonoclonals or the polyclonal antisera which contain the immunologicallysignificant portion can be used as antagonists, as well as the intactantibodies. Use of immunologically reactive fragments, such as the Fab,Fab′, of F(ab′)₂ fragments is often preferable, especially in atherapeutic context, as these fragments are generally less immunogenicthan the whole immunoglobulin.

The antibodies or fragments may also be produced, using currenttechnology, by recombinant means. Regions that bind specifically to thedesired regions of the transporter can also be produced in the contextof chimeric or CDR grafted antibodies of multiple species origin.

As described below, anti-ENaC antibodies are useful as modulators ofENaC activity, are useful in immunoassays for detecting ENaCexpression/activity and for purifying wild-type and altered variants ofthe ENaC proteins.

C. ENaC Encoding Nucleic Acid Molecules

As described above, the present invention is based, in part, onisolating nucleic acid molecules from humans that encode wild-type oraltered variants of the ENaC proteins. Accordingly, the presentinvention further provides nucleic acid molecules that encode the hereindisclosed wild-type and altered variants of the ENaC protein, as hereindefined, preferably in isolated form. For convenience, all ENaC encodingnucleic acid molecules will be referred to as ENaC encoding nucleic acidmolecules, the ENaC genes, or ENaC. The nucleotide sequence ofidentified wild-type and altered ENaC encoding nucleic acid moleculesare provided in FIG. 2.

As used herein, a “nucleic acid molecule” is defined as an RNA or DNAmolecule that encodes a peptide as defined above, or is complementary toa nucleic acid sequence encoding such peptides. Particularly preferrednucleic acid molecules will have a nucleotide sequence identical to orcomplementary to the human cDNA sequences herein disclosed. Specificallycontemplated are genomic DNA, cDNA, mRNA and antisense molecules, aswell as nucleic acids based on an alternative backbone or includingalternative bases, whether derived from natural sources or synthesized.Such nucleic acid molecules, however, are defined further as being noveland unobvious over any prior art nucleic acid molecules encodingnon-human homologues of ENaC isolated from non-human organisms and knownhuman ENaC proteins.

As used herein, a nucleic acid molecule is said to be “isolated” whenthe nucleic acid molecule is substantially separated from contaminantnucleic acid molecules that encode polypeptides other than ENaC. Askilled artisan can readily employ nucleic acid isolation procedures toobtain an isolated ENaC encoding nucleic acid molecule.

The present invention further provides fragments of the ENaC encodingnucleic acid molecules of the present invention. As used herein, afragment of an ENaC encoding nucleic acid molecule refers to a smallportion of the entire protein encoding sequence. The size of thefragment will be determined by its intended use. For example, if thefragment is chosen so as to encode an active portion of the ENaCprotein, such an intracellular or extracellular domain, then thefragment will need to be large enough to encode the functional region(s)of the ENaC protein. If the fragment is to be used as a nucleic acidprobe or PCR primer, then the fragment length is chosen so as to obtaina relatively small number of false positives during probing/priming.Table 2 identifies fragments of the ENaC genes that are particularlyuseful as selective hybridization probes or PCR primers. Such fragmentscontain regions that are conserved among wild-type or altered variantsof ENaC, regions of homology that are shared with the previouslyidentified ENaC genes, and regions that are altered in altered variantsof the ENaC genes.

Fragments of the ENaC encoding nucleic acid molecules of the presentinvention (i.e., synthetic oligonucleotides) that are used as probes orspecific primers for the polymerase chain reaction (PCR), or tosynthesize gene sequences encoding ENaC proteins, can easily besynthesized by chemical techniques, for example, the phosphotriestermethod of Matteucci, et al., J Am Chem Soc (1981) 103:3185-3191 or usingautomated synthesis methods. In addition, larger DNA segments canreadily be prepared by well known methods, such as synthesis of a groupof oligonucleotides that define various modular segments of the ENaCgene, followed by ligation of oligonucleotides to build the completemodified ENaC gene.

The ENaC encoding nucleic acid molecules of the present invention mayfurther be modified so as to contain a detectable label for diagnosticand probe purposes. As described above, such probes can be used toidentify nucleic acid molecules encoding other allelic variants ofwild-type or altered ENaC proteins and as described below, such probescan be used to diagnosis the presence of an altered variant of an ENaCprotein as a means for diagnosing a pathological condition caused byabnormal ion transport. A variety of such labels are known in the artand can readily be employed with the ENaC encoding molecules hereindescribed. Suitable labels include, but are not limited to, biotin,radiolabeled nucleotides, biotin, and the like. A skilled artisan canemploy any of the art known labels to obtain a labeled ENaC encodingnucleic acid molecule.

D. Isolation of Other Wild-Type and Altered Forms of ENaC EncodingNucleic Acid Molecules

As described above, the identification of the role of the ENaC proteinsin the pathology/severity of ion transport mediated deficiencies,particularly PHA1, has made possible the identification of severalaltered variants of the ENaC proteins that confer a pathology associatedwith abnormal (decreased) ion transport. These observations allow askilled artisan to isolate nucleic acid molecules that encode otherwild-type and altered variants of the ENaC proteins, in addition to thesequence herein described.

Essentially, a skilled artisan can readily use the amino acid sequenceof the human ENaC protein to generate antibody probes to screenexpression libraries prepared from cells. Typically, polyclonalantiserum from mammals such as rabbits immunized with the purifiedprotein (as described below) or monoclonal antibodies can be used toprobe a human cDNA or genomic expression library, such as lambda gtlllibrary, prepared from a normal or effected individual, to obtain theappropriate coding sequence for wild-type or altered variants of theENaC protein. The cloned cDNA sequence can be expressed as a fusionprotein, expressed directly using its own control sequences, orexpressed by constructions using control sequences appropriate to theparticular host used for expression of the enzyme. FIG. 3 identifiesimportant operative domains and domains that have been shown to containalterations in mutated variants of the ENaC protein. Such regions arepreferred sources of antigenic portions of the ENaC protein for theproduction of probe, diagnostic, and therapeutic antibodies.

Alternatively, a portion of the ENaC encoding sequence herein describedcan be synthesized and used as a probe to retrieve DNA encoding a memberof the ENaC family of proteins from individuals that have normal iontransport or from individuals suffering from a pathological conditionthat is a result of abnormal ion transport. Oligomers containingapproximately 18-20 nucleotides (encoding about a 6-7 amino acidstretch) are prepared and used to screen genomic DNA or cDNA librariesto obtain hybridization under stringent conditions or conditions ofsufficient stringency to eliminate an undue level of false positives.This method can be used to identify and isolate altered and wild-typevariants of the ENaC encoding sequences.

Additionally, pairs of oligonucleotide primers can be prepared for usein a polymerase chain reaction (PCR) to selectively amplify/clone anENaC-encoding nucleic acid molecule, or fragment thereof. A PCRdenature/anneal/extend cycle for using such PCR primers is well known inthe art and can readily be adapted for use in isolating other ENaCencoding nucleic acid molecules. Table 2 identifies regions of the humanENaC gene that are particularly well suited for use as a probe or asprimers. In general; the preferred primers will flank one or more exonsof the ENaC encoding nucleic acid molecule.

E. Methods for Identifying Pathological Conditions Involving AbnormalIon Transport

The present invention further provides methods for identifying cells andindividuals expressing active and altered variants of the ENaC proteinor the ENaC gene. Such methods can be used to diagnose biological andpathological processes associated with altered (decreased) iontransport, particularly PHA1, the progression of PHA1, thesusceptibility of PHA1 to treatment and the effectiveness of treatmentfor PHA1. The methods of the present invention are particularly usefulin identifying carriers of ion transport deficiencies, particularlyPHA1s, as well as in differentiating between PHA1 and other iontransport disorders. Specifically, the presence of wild-type or alteredvariants of an ENaC protein can be identified by determining whether awild-type or altered variant of the ENaC protein, or nucleic acidencoding the ENaC protein, is expressed in a cell. The expression of analtered variant, or departure from the normal level of ENaC expression,(decrease in expression) can be used as a means for diagnosingpathological conditions mediated by abnormal ENaC activity/expression,differentiating between various ion transport deficiencies, and foridentifying carriers of an ion transport deficiency.

A variety of immunological and molecular genetic techniques can be usedto determine if a wild-type or an altered variant of an ENaC protein isexpressed/produced in a particular cell and/or the level at which theprotein is expressed. The preferred methods will identify whether awild-type or mutated from of the ENaC protein is expressed.

In general, an extract containing nucleic acid molecules or an extractcontaining proteins is prepared from cells of an individual. The extractis then assayed to determine whether an ENaC protein, or an ENaCencoding nucleic acid molecule, is produced in the cell. The type ofprotein/nucleic acid molecule expressed and/or the degree/level ofexpression, provides a measurement of the nature and degree of ENaCactivity.

For example, to perform a diagnostic test based on nucleic acidmolecules, a suitable nucleic acid sample is obtained and prepared froma subject using conventional techniques. DNA can be prepared, forexample, simply by boiling a sample in SDS. Most typically, for nucleicacid samples, a blood sample, a buccal swab, a hair follicle preparationor a nasal aspirate is used as a source of cells to provide the nucleicacid molecules. The extracted nucleic acid can then be subjected toamplification, for example by using the polymerase chain reaction (PCR)according to standard procedures, to selectively amplify an ENaCencoding nucleic acid molecule or fragment thereof. The size of theamplified fragment (typically following restriction endonucleasedigestion) is then determined using gel electrophoresis or thenucleotide sequence of the fragment is determined (for example, seeWeber and May Am J Hum Genet (1 989) 44:388-339; Davies, J. et al.Nature (1994) 371:130-136)). The resulting size of the fragment orsequence is then compared to the known wild-type, predicted wild-type,known altered variants and predicted altered variants of the ENaCprotein. Using this method, the presence of wild-type or alteredvariants of an ENaC protein can be differentiated and identified.

Alternatively, the presence or absence of one or more single base-pairpolymorphism(s) within an ENaC encoding nucleic acid molecule can bedetermined by conventional methods which included, but are not limitedto, manual and automated fluorescent DNA sequencing, selectivehybridization probes, primer extension methods (Nikiforov, T. T. et al.Nucl Acids Res (1994) 22:4167-4175); oligonucleotide ligation assay(OLA) (Nickerson, D. A. et al. Proc Natl Acad Sci USA (1990)87:8923-8927); allele-specific PCR methods (Rust, S. et al. Nucl AcidsRes (1993) 6:3623-3629); RNase mismatch cleavage, single strandconformation polymorphism (SSCP) (Orita, M. et al., Proc Natl Acad SciUSA 86:2766-2770 (1989)), denaturing gradient gel electrophoresis (DGGE)and the like. The present diagnosis method is particularly well suit foruse in biochip technologies that are being developed to be used toidentify whether one of many sequence variations is present in a sample.A skilled artisan can readily adapt any nucleic acid analytical methodfor use in determining whether a sample contains nucleic acid moleculesthat encode a wild-type or altered variant of an ENaC protein.

To perform a diagnostic test based on proteins, a suitable proteinsample is obtained and prepared from a subject using conventionaltechniques. Protein samples can be prepared, for example, simply bymixing the sample with SDS followed by salt precipitation of a proteinfraction. Typically, for protein samples, a blood sample, a buccal swab,a nasal aspirate, or a biopsy of cells from tissues expressing an ENaCprotein is used as a source of cells to provide the protein molecules.The extracted protein can then be analyzed to determine the presence ofa wild-type or altered variant of an ENaC protein using known methods.For example, the presence of specific sized or charged variants of aprotein can be identified using mobility in an electric filed.Alternatively, wild-type or altered variant specific antibodies can beused. A skilled artisan can readily adapt known protein analyticalmethods to determine if a sample contains a wild-type and/or alteredvariant of an ENaC protein.

EnaC expression can also be used in methods to identify disorders thatoccur as a result of a decrease in the expression of a naturallyoccurring ENaC gene. Specifically, nucleic acid probes that detect. mRNAcan be used to detect cells or tissues that express an ENaC protein andthe level of such expression.

The presence of only an altered variant of an ENaC protein (homozygousstate) in a sample is diagnostic of PHA1. Altered variants of the ENaCprotein, when present in sample that additionally contains a wild-typevariant of ENaC (heterozygous state), is diagnostic for carriers of PHA1and individuals expressing lower levels of active ENaC. Decreased ENaCactivity leads to minor ion transport deficiencies and a susceptibilityto adverse drug reaction.

Alternatively, ENaC expression can also be used in methods to identifyagents that increase or decrease the level of expression of a naturallyoccurring ENaC gene. For example, cells or tissues expressing an ENaCprotein can be contacted with a test agent to determine the effects ofthe agent on ENaC expression. Agents that activate ENaC expression canbe used as an agonist of ENaC activity whereas agents that decrease ENaCexpression can be used as an antagonist of ENaC activity.

F. rDNA Molecules Containing an ENaC Encoding Nucleic Acid Molecule

The present invention further provides recombinant DNA molecules (rDNAs)that contain one or more of the wild-type or altered ENaC encodingsequences herein described, or a fragment of the herein-describednucleic acid molecules. As used herein, an rDNA molecule is a DNAmolecule that has been subjected to molecular manipulation in vitro.Methods for generating rDNA molecules are well known in the art, forexample, see Sambrook et al., Molecular Cloning (1989). In the preferredrDNA molecules, an ENaC encoding DNA sequence that encodes a wild-typeor altered variant of the ENaC protein is operably linked to one or moreexpression control sequences and/or vector sequences. Most preferably,the ENaC encoding nucleic acid molecules will encode one of the novelaltered variants herein described.

The choice of vector and/or expression control sequences to which one ofthe ENaC encoding sequences of the present invention is operably linkeddepends directly, as is well known in the art, on the functionalproperties desired, e.g., protein expression, and the host cell to betransformed. A vector contemplated by the present invention is at leastcapable of directing the replication or insertion into the hostchromosome, and preferably also expression, of an ENaC encoding sequenceincluded in the rDNA molecule.

Expression control elements that are used for regulating the expressionof an operably linked protein encoding sequence are known in the art andinclude, but are not limited to, inducible promoters, constitutivepromoters, secretion signals, enhancers, transcription terminators andother regulatory elements. Preferably, an inducible promoter that isreadily controlled, such as being responsive to a nutrient in the hostcell's medium, is used.

In one embodiment, the vector containing an ENaC encoding nucleic acidmolecule will include a prokaryotic replicon, i.e., a DNA sequencehaving the ability to direct autonomous replication and maintenance ofthe recombinant DNA molecule intrachromosomally in a prokaryotic hostcell, such as a bacterial host cell, transformed therewith. Suchreplicons are well known in the art. In addition, vectors that include aprokaryotic replicon may also include a gene whose expression confers adetectable marker such as a drug resistance. Typical bacterial drugresistance genes are those that confer resistance to ampicillin ortetracycline.

Vectors that include a prokaryotic replicon can further include aprokaryotic or viral promoter capable of directing the expression(transcription and translation) of the ENaC encoding sequence in abacterial host cell, such as E. coli. A promoter is an expressioncontrol element formed by a DNA sequence that permits binding of RNApolymerase and transcription to occur. Promoter sequences compatiblewith bacterial hosts are typically provided in plasmid vectorscontaining convenient restriction sites for insertion of a DNA segmentof the present invention. Typical of such vector plasmids are pUC8,pUC9, pBR322 and pBR329 available from Biorad Laboratories (Richmond,Calif.), pPL and pKK223 available from Pharmacia, Piscataway, N.J.

Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can also be used to variant rDNAmolecules that contain an ENaC encoding sequence. Eukaryotic cellexpression vectors are well known in the art and are available fromseveral commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desired DNAsegment. Typical of such vectors are PSVL and pKSV-10 (Pharmacia),pBPV-1/pML2d (International Biotechnologies, Inc.), pTDT1 (ATCC,#31255), the vector pCDM8 described herein, and the like eukaryoticexpression vectors.

Eukaryotic cell expression vectors used to construct the rDNA moleculesof the present invention may further include a selectable marker that iseffective in an eukaryotic cell, preferably a drug resistance selectionmarker. A preferred drug resistance marker is the gene whose expressionresults in neomycin resistance, i.e., the neomycin phosphotransferase(neo) gene. Southern et al., J Mol Anal Genet (1982)1:327-341.Alternatively, the selectable marker can be present on a separateplasmid, and the two vectors are introduced by cotransfection of thehost cell, and selected by culturing in the presence of the appropriatedrug for the selectable marker.

G. Host Cells Containing an Exogenously Supplied ENaC Encoding NucleicAcid Molecule

The present invention further provides host cells transformed with anucleic acid molecule that encodes one of the human wild-type or alteredENaC protein of the present invention. The host cell can be eitherprokaryotic or eukaryotic. Eukaryotic cells useful for expression of anENaC protein are not limited, so long as the cell line is compatiblewith cell culture methods and compatible with the propagation of theexpression vector and expression of an ENaC gene. Preferred eukaryotichost cells include, but are not limited to, yeast, insect and mammaliancells, preferably vertebrate cells such as those from a mouse, rat,monkey or human fibroblastic cell line, the most preferred being cellsthat do not naturally express a human ENaC protein.

Any prokaryotic host can be used to express an ENaC-encoding rDNAmolecule. The preferred prokaryotic host is E. coli.

Transformation of appropriate cell hosts with an rDNA molecule of thepresent invention is accomplished by well known methods that typicallydepend on the type of vector used and host system employed. With regardto transformation of prokaryotic host cells, electroporation and salttreatment methods are typically employed, see, for example, Cohen etal., Proc Acad Sci USA (1972) 69:2110; and Maniatis et al., MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1982). With regard to transformation of vertebrate cellswith vectors containing rDNAs, electroporation, cationic lipid or salttreatment methods are typically employed, see, for example, Graham etal., Virol (1973) 52:456; Wigler et al., Proc Natl Acad Sci USA (1979)76:1373-76.

Successfully transformed cells, i.e., cells that contain an rDNAmolecule of the present invention, can be identified by well knowntechniques. For example, cells resulting from the introduction of anrDNA of the present invention can be cloned to produce single colonies.Cells from those colonies can be harvested, lysed and their DNA contentexamined for the presence of the rDNA using a method such as thatdescribed by Southern, J Mol Biol (1975) 98:503, or Berent et al.,Biotech (1985) 3:208 or the proteins produced from the cell assayed viaan immunological method.

H. Production of an ENaC Protein Using an rDNA Molecule

The present invention further provides methods for producing a humanwild-type or altered ENaC protein that uses one of the ENaC encodingnucleic acid molecules herein described. In general terms, theproduction of a recombinant human wild-type or altered ENaC proteintypically involves the following steps.

First, a nucleic acid molecule is obtained that encodes an ENaC protein,such as the nucleic acid molecule depicted in FIG. 2. If the ENaCencoding sequence uninterrupted by introns, it is directly suitable forexpression I any host. If not, then a spliced variant of the ENaCencoding nucleic acid molecule can be generated and used or the introncontaining nucleic acid molecule can be used in a compatible eukaryoticexpression system.

The ENaC encoding nucleic acid molecule is then preferably placed in anoperable linkage with suitable control sequences, as described above, tovariant an expression unit containing the ENaC encoding sequence. Theexpression unit is used to transform a suitable host and the transformedhost is cultured under conditions that allow the production of the ENaCprotein. Optionally the ENaC protein is isolated from the medium or fromthe cells; recovery and purification of the protein may not be necessaryin some instances where some impurities may be tolerated.

Each of the foregoing steps can be done in a variety of ways. Forexample, the desired coding sequences may be obtained from genomicfragments and used directly in an appropriate host. The construction ofexpression vectors that are operable in a variety of hosts isaccomplished using an appropriate combination of replicons and controlsequences. The control sequences, expression vectors, and transformationmethods are dependent on the type of host cell used to express the geneand were discussed in detail earlier. Suitable restriction sites can, ifnot normally available, be added to the ends of the coding sequence soas to provide an excisable gene to insert into these vectors. A skilledartisan can readily adapt any host/expression system known in the artfor use with ENaC encoding sequences to produce an ENaC protein.Particularly well suited are expression systems that result in theproduction of lipid vesicles containing the expressed protein. Suchlipid containing vesicles are well suited for identifying agonists andantagonists of the ENaC protein.

I. Ion Transport

As provided above, alterations in the ENaC protein cause pathologicalconditions that are a result of abnormal ion transport. Accordingly, thewild-type and altered variants of the ENaC proteins of the presentinvention can be used in methods to alter the extra or intracellularconcentration of sodium. In general, the extra or intracellularconcentration of sodium can be altered by altering the expression of anENaC protein or the activity of an ENaC protein.

There are a number of situation in which it is desirable to alter theextra or intracellular concentration of sodium. Abnormal extra orintracellular sodium leads to salt wasting and hypokalaemic acidosis.Hence, an ENaC protein, or ENaC gene, can be used as a target for, or asmeans to alter extra or intracellular sodium concentration. For example,an ENaC gene can be introduced and expressed in cells to increase ENaCexpression. This provides a means and methods for altering extra andintracellular ion levels.

There are pathological conditions characterized by inappropriate extraor intracellular sodium concentrations. For example, PHA1, hypokalaemicacidosis and salt wasting are all associated with abnormal intracellularor extracellular sodium concentration. Accordingly, ENaCactivity/expression is targeted as a means of treating these conditions.Various methods for regulating ENaC activity/expression are discussed indetail below.

J. Identification of Agents that Bind to an ENaC Protein

Another embodiment of the present invention provides methods foridentifying agents that are agonists or antagonists of the ENaC proteinsherein described. Specifically, agonists and antagonists of an ENaCprotein can be first identified by the ability of the agent to bind toone of the wild-type or altered variants of the ENaC protein hereindescribed. Agents that bind to an ENaC protein can then be tested forthe ability to stimulate or block sodium transport in an ENaC expressingcell.

In detail, an ENaC protein is mixed with an agent. After mixing underconditions that allow association of ENaC with the agent, the mixture isanalyzed to determine if the agent bound the ENaC protein. Agonists andantagonists are identified as being able to bind to an ENaC protein,

The ENaC protein used in the above assay can be: an isolated and fullycharacterized protein, a partially purified protein, a cell that hasbeen altered to express an ENaC protein or a fraction of a cell that hasbeen altered to express an ENaC protein. Further, the ENaC protein canbe the entire ENaC protein, a specific fragment of the ENaC protein or asingle subunit of the ENaC protein. It will be apparent to one ofordinary skill in the art that so long as the ENaC protein can beassayed for agent binding, e.g., by a shift in molecular weight orchange in cellular ion content, the present assay can be used.

The method used to identify whether an agent binds to an ENaC proteinwill be based primarily on the nature of the ENaC protein used. Forexample, a gel retardation assay can be used to determine whether anagent binds to a soluble fragment of an ENaC protein whereas patchclamping, voltage clamping, ion-sensitive microprobes or ion-sensitivechromaphores can be used to determine whether an agent binds to a cellexpressing an ENaC protein and affects the activity of the expressedprotein. A skilled artisan can readily employ numerous techniques fordetermining whether a particular agent binds to an ENaC protein.

Once binding is demonstrated, the agent can be further tested for theability to modulate the activity of a wild-type or altered variant ofthe ENaC protein using a cell or oocyte expression system and an assaythat detects ENaC activity. For example, voltage or patch clamping,ion-sensitive microprobes or ion-sensitive chromaphores and expressionin Xenopus oocytes or recombinant host cells can be used to determinewhether an agent that binds an ENaC protein can agonize or antagonizeENaC activity.

As used herein, an agent is said to antagonize ENaC activity when theagent reduces ENaC activity. The preferred antagonist will selectivelyantagonize ENaC, not affecting any other cellular proteins, particularlyother ion transport proteins. Further, the preferred antagonist willreduce ENaC activity by more than 50%, more preferably by more than 90%,most preferably eliminating all ENaC activity.

As used herein, an agent is said to agonize ENaC activity when the agentincreases ENaC activity. The preferred agonist will selectively agonizealtered variants of ENaC, not affecting any other cellular proteins,particularly other ion transport proteins. Further, the preferredagonist will increase ENaC activity by more than 50%, more preferably bymore than 90%, most preferably more than doubling the level of ENaCactivity.

Agents that are assayed in the above method can be randomly selected orrationally selected or designed. As used herein, an agent is said to berandomly selected when the agent is chosen randomly without consideringthe specific sequences of the ENaC protein. An example of randomlyselected agents is the use a chemical library or a peptide combinatoriallibrary, or a growth broth of an organism.

As used herein, an agent is said to be rationally selected or designedwhen the agent is chosen on a nonrandom basis which takes into accountthe sequence of the target site and/or its conformation in connectionwith the agent's action. Agents can be rationally selected or rationallydesigned by utilizing the peptide sequences that make up the ENaCprotein. For example, a rationally selected peptide agent can be apeptide whose amino acid sequence is identical to a fragment of an ENaCprotein.

The agents of the present invention can be, as examples, peptides, smallmolecules, and vitamin derivatives, as well as carbohydrates. A skilledartisan can readily recognize that there is no limit as to thestructural nature of the agents of the present invention. One class ofagents of the present invention are peptide agents whose amino acidsequences are chosen based on the amino acid sequence of the ENaCprotein.

The peptide agents of the invention can be prepared using standard solidphase (or solution phase) peptide synthesis methods, as is known in theart. In addition, the DNA encoding these peptides may be synthesizedusing commercially available oligonucleotide synthesis instrumentationand produced recombinantly using standard recombinant productionsystems. The production using solid phase peptide synthesis isnecessitated if non-gene-encoded amino acids are to be included.

Another class of agents of the present invention are antibodiesimmunoreactive with critical positions of the ENaC protein. As describedabove, antibodies are obtained by immunization of suitable mammaliansubjects with peptides, containing as antigenic regions, those portionsof the ENaC protein intended to be targeted by the antibodies Criticalregions include the domains identified in FIG. 3.

K. Uses of Agents that Bind to an ENaC Protein

As provided in the Background section, the ENaC proteins are involved inregulating intracellular and extracellular sodium concentration. Agentsthat bind an ENaC protein and act as an agonist or antagonist can beused to modulate biological and pathologic processes associated withENaC function and activity. In detail, a biological or pathologicalprocess mediated by ENaC can be modulated by administering to a subjectan agent that binds to an ENaC protein and acts as an agonist orantagonist of ENaC activity.

As used herein, a subject can be any mammal, so long as the mammal is inneed of modulation of a pathological or biological process mediated byENaC. The term “mammal” means an individual belonging to the classMammalia. The invention is particularly useful in the treatment of humansubjects.

As used herein, a biological or pathological process mediated by ENaCrefers to the wide variety of cellular events mediated by an ENaCprotein. Pathological processes refer to a category of biologicalprocesses which produce a deleterious effect. For example, pathologicalprocesses mediated by ENaC include PHA 1, hypokalaemic acidosis and saltwasting. These pathological processes can be modulated using agents thatreduce or increase the activity of an ENaC protein. Preferably, theagent will act to activate an otherwise inactive altered variant of anENaC protein.

As used herein, an agent is said to modulate a pathological process whenthe agent reduces the degree or severity of the pathological process.For example, an agent is said to modulate PHA1 when the agentcontributes to normal intra and extracellular sodium concentrations.

L. Administration of Agonists and Antagonists of an ENaC Protein

Agonists and antagonists of the ENaC protein can be administered viaparenteral, subcutaneous, intravenous, intramuscular, intraperitoneal,transdermal, or buccal routes. Alternatively, or concurrently,administration may be by the oral route. The dosage administered will bedependent upon the age, health, and weight of the recipient, kind ofconcurrent treatment, if any, frequency of treatment, and the nature ofthe effect desired. For example, to treat pathological conditionsresulting from abnormal ion transport, such as water retention,increased blood pressure, chronic respiratory and metabolic acidosis,inflammation, etc., an agent that modulates ENaC activity isadministered systemically or locally to the individual being treated. Asdescribed below, there are many methods that can readily be adapted toadminister such agents.

The present invention further provides compositions containing anantagonist or agonist of an ENaC protein that is identified by themethods herein described. While individual needs vary, a determinationof optimal ranges of effective amounts of each component is within theskill of the art. Typical dosages comprise 0.1 to 100 μg/kg body wt. Thepreferred dosages comprise 0.1 to 10 μg/kg body wt. The most preferreddosages comprise 0.1 to 1 μg/kg body wt.

In addition to the pharmacologically active agent, the compositions ofthe present invention may contain suitable pharmaceutically acceptablecarriers comprising excipients and auxiliaries which facilitateprocessing of the active compounds into preparations which can be usedpharmaceutically for delivery to the site of action. Suitableformulations for parenteral administration include aqueous solutions ofthe active compounds in water-soluble variant, for example,water-soluble salts. In addition, suspensions of the active compoundsand as appropriate, oily injection suspensions may be administered.Suitable lipophilic solvents or vehicles include fatty oils, forexample, sesame oil, or synthetic fatty acid esters, for example, ethyloleate or triglycerides. Aqueous injection suspensions may containsubstances which increase the viscosity of the suspension and include,for example, sodium carboxymethyl cellulose, sorbitol, and/or dintran.Optionally, the suspension may also contain stabilizers. Liposomes canalso be used to encapsulate the agent for delivery into the cell.

The pharmaceutical formulation for systemic administration according tothe invention may be formulated for enteral, parenteral or topicaladministration. Indeed, all three types of formulations may be usedsimultaneously to achieve systemic administration of the activeingredient.

Suitable formulations for oral administration include hard or softgelatin capsules, pills, tablets, including coated tablets, elixirs,suspensions, syrups or inhalations and controlled release variantsthereof.

M. Combination Therapy

The agents of the present invention that modulate ENaC activity can beprovided alone, or in combination with another agents that modulate aparticular biological or pathological process. For example, an agent ofthe present invention that reduces ENaC activity can be administered incombination with other agents that affect the sodium transport. As usedherein, two agents are said to be administered in combination when thetwo agents are administered simultaneously or are administeredindependently in a fashion such that the agents will act at the sametime.

N. Animal Models and Gene Therapy

The ENaC genes and the ENaC proteins can also serve as targets for genetherapy in a variety of contexts. For example, in one application,ENaC-deficient non-human animals can be generated using standardknock-out procedures to inactivate an ENaC gene or, if such animals arenon-viable, inducible ENaC antisense molecules can be used to regulateENaC activity/expression. Alternatively, an animal can be altered so asto contain an ENaC or antisense-ENaC expression unit that directs theexpression of ENaC or the antisense molecule in a tissue specificfashion. In such uses, a non-human mammal, for example a mouse or a rat,is generated in which the expression of the ENaC gene is altered byinactivation or activation. This can be accomplished using a variety ofart-known procedures such as targeted recombination. Once generated, theENaC-deficient animal, the animal that expresses ENaC in a tissuespecific manner, or an animal that expresses an antisense molecule canbe used to 1) identify biological and pathological processes mediated byENaC, 2) identify proteins and other genes that interact with ENaC, 3)identify agents that can be exogenously supplied to overcome ENaCdeficiency and 4) serve as an appropriate screen for identifyingmutations within ENaC that increase or decrease activity.

For example, it is possible to generate transgenic mice expressing thehuman minigene for ENaC in a tissue specific-fashion and test the effectof over-expression of the protein in cells and tissues that normally donot contain ENaC. This strategy has been successfully used for otherproteins, namely bcl-2 (Veis et al. Cell 75:229 (1993)). Such anapproach can readily be applied to the ENaC protein and can be used toaddress the issue of a potential beneficial effect of ENaC in a specifictissue area.

In another embodiment, genetic therapy can be used as a means formodulating an ENaC-mediated biological or pathological processes. Forexample, it may be desirable to introduce into a subject being treated agenetic expression unit that encodes a modulator of ENaC expression,such as an antisense encoding nucleic acid molecule or an ENaC encodingnucleic acid molecule, or a functional ENaC expression unit. Suchmodulators can either be constitutively produced or inducible within acell or specific target cell. This allows a continual or induciblesupply of a modulator of ENaC or the protein expression within asubject.

The following examples are intended to illustrate, but not to limit,aspects of the present invention.

EXAMPLE 1 Methods

Genotyping and SSCP

SSCP of all coding exons of α, β, and γENaC was performed using specificprimers to amplify exons or exon fragments of exons 150-250 base pairsin length from genomic DNA by PCR as previously described (Shimkets, R.A. et al. Cell 79:407-414 (1994)). Forty-three sets of primers were used(Table 2), based on the cloning and characterization of the intron-exonorganization of each genomic locus (McDonald, F. J., et al. Am. J.Physiol. 268:L728-734 (1994); McDonald, F. J., et al. Am. J. Physiol.268: C1157-C1163 (1995); Lu et al., in preparation). Primers are inintrons with the exception of large coding exons in which overlappingprimer sets in exons are employed. PCR was performed using specificprimers and genomic DNA as template, and products were fractionated onnon-denaturing gels as described previously (Simon, D. et al. NatureGenet. 12:24-30 (1996)). Novel SSCP conformers were identified byautoradiography, purified, and subjected to direct DNA sequence analysisas described previously (380. In all cases, DNA sequences were confirmedby sequencing both DNA strands. Genotypes of markers closely linked to aor β-γENaC were determined by polymerase chain reaction using specificprimers and genomic DNA as template by standard methods. Markers tightlylinked to the β-γENaC locus were genotyped as previously described(Shimkets, R. A. et al. Cell 79:407-414 (1994)). Marker loci linked toαENaC were identified by use of an RFLP detected by hybridizing ratαENaC cDNA to TaqI-cut human genomic DNA. Genotyping of 166 individualsin CEPH kindreds revealed linkage of αENaC to loci D12S314 and D12S93(lod score of 8.3 for linkage to D12S314 at a recombination fraction of4%), with a peak multipoint lod score localizing the gene 2 cM telomericto D12S314. Genomic DNA of subjects from PHA kindreds was prepared fromvenous blood by standard methods (Bell, G., et al. Proc. Natn. Acad.Sci. USA 78:5759-5763 (1981)).

Construction of rat βENaC37S

Serine was substituted for glycine at residue 37 of rat βENaC cDNA bysite-directed mutagenesis using a mutagenic primer and PCR. PCR wasperformed using rat βENaC cDNA21 as a template, a sense mutagenic primer(CCAACACACACAGCCCCAAAC) (SEQ. ID NO:19) extending from nucleotide 170 to190 (codons 33-39) of the βENaC cDNA sequence and altering nucleotide181 from G to A, and a reverse or antisense primer(CTTGACCTTGGAGTACTGGAAG)(SEQ. ID NO:20), extending from nucleotide 378to 400. After PCR, this product was purified and used as a primer inconjunction with the vector Sp6 primer to direct PCR using the rat βENaCcDNA as a template. The resulting product contained the desiredmutation, and was cleaved at a unique EcoRI cleavage site in vectorsequence and a unique ScaI site in codon 146. This fragment was purifiedand substituted for the corresponding wild-type sequence in the βENaCcDNA. The structure and sequence of the resulting mutant construct wasconfirmed by DNA sequencing.

Expression Studies of Normal and Mutant ENaC

Complementary RNAs (cRNA) of each α, β and γ subunit were synthesized invitro. Equal saturating concentrations of each subunit cRNA (3 ng totalcRNA of each subunit/oocyte) were injected into stage V to VI oocytes aspreviously described (Schild, L. et al. Proc. Natn. Acad. Sci. USA92:5699-5703 (1995)); cRNAs injected together were normal a, β and γsubunits; normal α and γ subunits plus mutant β subunits; normal α and γsubunits alone with no β subunits. Oocytes from the same frog wereinjected on the same day with wild-type or mutant constructs.Twenty-four hours after injection, whole-oocyte currents were measuredusing two-electrode voltage clamp technique in a medium containing: 120mM NaCl, 2.5 mM KCl, 1.8 mM CaCl₂, 10 mM HEPES-NaOH pH 7.2. Theexpressed ENaC channel activity was assessed by measurement of theamiloride-sensitive Na current, defined as the difference between the Nacurrent recorded at a membrane potential of −100 mV in the absence andpresence in the medium of 5 μM amiloride. The results were analyzed byT-test.

Comparable expression-levels of the wild-type β subunit and onecontaining the G37S mutation in Xenopus oocytes were ensured byimmunoprecipitation. Oocytes injected with cRNAs encoding either α, βand γ, or α and γ or α, βG37S and γ subunits were labeled for 14 h with(35S)methionine, and microsomal membranes were prepared. The threesubunits were immunoprecipitated under denaturing conditions withspecific antisera and immunoprecipitates were separated on a 8%SDS-polyacrylamide gel (Duc, C., et al. J. Cell. Biol.127:1907-1921(1994)).

Results

PHA Kindreds

Seven PHA1 kindreds containing 10 living affected subjects wereascertained in Saudi Arabia and Israel (Table 1). Two of these kindreds,PHA K10, and PHA K3 have been previously reported (Mathew, P. M., et al.Clinical Pediatrics. 1:58-60 (1993); Hanukoglu, A. J. Clin. Endocrin. &Metab. 73,936-944 (1991)). All affected subjects were the product ofconsanguineous union, supporting autosomal recessive transmission (FIG.1). Most subjects were diagnosed in the neonatal period, and all hadclinical features of severe dehydration, hypotension, hyponatremia,hyperkalaemia, and metabolic acidosis despite normal glomerularfiltration rate. Plasma renin activity and aldosterone concentrationswere markedly elevated. No subjects had signs of abnormal virilization.Multi-organ involvement was documented in PHA K3 (Hanukoglu, A. J. Clin.Endocrin. & Metab. 73,936-944 (1991)). Several index cases had siblingswho died with a similar syndrome in the first days of life (FIG. 1).Clinical management consisted of dietary sodium supplementation and useof an ion binding resin or dialysis to reduce potassium levels. Theconstellation of clinical features permitted definitive diagnosis ofPHA1 in all affected subjects.

Mutations in αENaC in PHA1

The a subunit of ENaC is required for ENaC activity (Canessa, C. M. etal. Nature 367:463-467 (1994)), and consequently loss of functionmutations in this gene could result in a syndrome similar to PHA1.Affected subjects arising from consanguineous union are expected to behomozygous for the same mutant allele at the trait locus; in contrast,random loci will be homozygous for an ancestral allele with likelihood 1in 16 in the offspring of 1st cousins and 1 in 64 in the offspring ofsecond cousins, providing a powerful test of linkage (Lander, E. S. etal. Science 236:1567-1570 (1987)). Accordingly, the knowledge of theintron-exon structure of αENaC and single-strand conformationalpolymorphism (SSCP was used) to screen for molecular variants in exonsand intron-exon boundaries of this gene in PHA1 patients. Affectedsubjects in 4 of the 7 kindreds showed novel αENaC variants that in eachcase were homozygous in all affected subjects in each kindred (FIGS. 2aand 2 b); no other missense variants or variants altering consensussplice sites were identified. In each case the parents were heterozygousfor these variants and none of the unaffected siblings inherited twocopies of the variant, demonstrating cosegregation of these variantswith PHA1 in these kindreds (FIGS. 1 and 2).

That these variants are homozygous by descent from a great-grandparentis supported by the finding that these variants are rare (absent in 160alleles from unrelated subjects who do not have PHA1) and that twohighly polymorphic loci tightly linked to αENaC, D12S314 and D12S93(Gyapay, G. et al. Nature Genet. 7:246-339 (1994)), are each homozygousin affected subjects of these kindreds (data not shown).

Affected subjects in three of these kindreds, all Saudi nativesascertained in Dhahran but not known to be related to one another,showed indistinguishable homozygous variants in exon 2 of αENaC (FIG.2a). DNA sequence analysis of the variant in these kindreds revealed a 2base pair deletion at codon I68, introducing a frameshift mutation (FIG.2a). This mutation disrupts the encoded protein prior to the firsttransmembrane domain (FIG. 3a); the encoded protein bears no similarityto the normal protein from amino acid 68 through amino acid 144, where atermination codon ends translation.

The DNA sequence of the homozygous αENaC variant in the fourth kindred,PHA K3 from Israel, reveals a single base substitution changing codonR508 from CGA to TGA and introducing a premature termination codon (FIG.2b, FIG. 3a). This codon is in the extracellular domain, and thusresults in a protein containing a normal first transmembrane domain,part of the extracellular domain and missing the second transmembranedomain as well as the intracytoplasmic C-terminus.

Both of these mutations result in αENaC subunits that lead to loss ofENaC channel activity since an intact second transmembrane domain isrequired for normal channel activity (Li, X. J., et al. MolecularPharmacology 47:1133-1140 (1995)). These mutations can thus explain thepathogenesis of PHA1 in these families.

Another mutation in the a subunit of EnaC, which also causespseudohypoaldosteronism type I, is the mutation of cysteine 133 totyrosine.

Mutation in βENaC in PHA1

Identification of mutations in αENaC in 4 PHA1 kindreds leaves open thequestion of whether other kindreds also harbor mutations at this locus,or alternatively whether there might be mutations at other loci thataccount for the disease in these remaining kindreds. The β and γsubunits of ENaC were tested for mutation by systematic screening ofexons of these genes.

This screening in all PHA1 kindreds revealed a single variant alteringthe encoded protein in PHA K8 (FIG. 2c). This kindred is particularlyinformative because two unaffected brothers had affected offspring, oneof these via union with a second cousin, the other via a spouse ofuncertain relationship (FIG. 1). The affected third cousins arehomozygous for the same variant, while none of their unaffected siblingsor relatives are homozygous for this variant; moreover, this variant isabsent in 160 alleles of unrelated healthy subjects. In addition,genotypes of marker loci tightly linked to bENAC, D16S412, D16S417 andD16S420 are all homozygous in these affected subjects but not theirunaffected relatives, strongly supporting the identity by descent of theobserved mutation.

DNA sequence analysis reveals that this βENaC variant substitutes serinefor glycine at amino acid 37 of βENaC (FIG. 2C, FIG. 3). While thecytoplasmic amino termini of α, β and γ ENaC generally show little aminoacid sequence identity with one another, it is noteworthy that G37 is ina segment that shows homology among all members of the extended ENaCfamily ranging from humans to C. elegans (FIG. 3b) (Canessa, C. M., etal. Nature 361:467-470 (1993); Canessa, C. M. et al. Nature 367:463-467(1994); McDonald, F. J., et al. Am. J. Physiol. 268:L728-734 (1994);McDonald, F. J., et al. Am. J. Physiol. 268:C1157-C1163 (1995); Puoti,A. et al. Am J. Physiol. 269:C188-C197 (1995); Waldmann, R., et al. J.of Biol. Chem. 270:27411-27414 (1995); Huang, M. et al. Nature367:467-470 (1994); Chalfie, M. et al. Nature 345:410-416 (1990)).

The functional significance of the G37S variant was assessed byexpression of this βENaC variant in conjunction with normal α and γsubunits in Xenopus oocytes as described previously (Hansson, J. H. etal. Nature Genetics 11:76-82 (1995); Hansson, J. H. et al. Proc. Natn.Acad. Sci. USA 92:11495-11499 (1995); Schild, L. et al. Proc. Natn.Acad. Sci. USA 92:5699-5703 (1995)). The amiloride-sensitive Na⁺current, measured by 2-electrode voltage clamp in oocytes expressing thewild-type ENaC, ENaC containing the mutant β subunit, and channelscontaining only α and γ subunits was determined and compared (FIG. 4).In order to compare levels of ENaC proteins in oocytes expressingwild-type and mutant channels, subunits were immunoprecipitated fromoocyte membranes using specific antibodies to each subunit (Duc, C., etal. J. Cell. Biol. 127:1907-1921 (1994)). The results demonstratedindistinguishable levels of each subunit in oocytes expressing wild typeand mutant ENaC (data not shown). Comparison of Na⁺ currents in oocytesexpressing wild-type or mutant ENaCs demonstrate a highly significantreduction in ENaC activity in oocytes expressing the mutant β subunit(40% of wild-type activity, p<0.00001). Oocytes expressing the mutant βsubunit, however, still have significantly higher activity than channelsexpressing no β subunit (p=0.00001), suggesting that this mutation doesnot result in complete loss of function.

The strong evidence of cosegregation of βENaC G37S with PHA in thiskindred and the loss of function demonstrated on expression indicatesthe functional significance of this mutation, revealing geneticheterogeneity of PHA1.

Another mutation in the β subunit of ENaC which also causespseudohypoaldosteronism type I occurs when glycine-37 is mutated to aserine residue.

TABLE 1 Characteristics of index cases of PHA 1 kindreds KindredLocation Ethnicity Age Na + K + PAC Mutation PHA K10 Saudi Arabia Saudi7 d 124 7.7 1.87 αENaC 168fr PHA K13 Saudi Arabia Saudi 1 d 126 11.26.28 αENaC 168fr PHA K14 Saudi Arabia Saudi 8 d 128 10.9 15.16 αENaC168fr PHA K3 Israel Iranian 9 d 125 10.0 14.27 αENaC R508stop Jew PHA K8Israel Arabic 19 d  133 8.2 1.00 βENaC G37S PHA K12 Saudi ArabiaPakistani 235 d  107 6.9 3.24 none PHA K7 Saudi Arabia Sudanese 5 d 11211.0 8.64 none Age, age at clinical presentation (days); Na⁺, serumsodium concentration (mM), normal 138-142; K⁺ serum potassiumconcentration (mM), normal 3.5-5.0; PAC, plasma aldosteroneconcentration (ng dl-1), normal 1-95. fr, frameshift.

TABLE 2 Primers used to amplify coding regions of ENaC subunits PrimerExon Forward Reverse A-1 1 ACCCTTGCTCTCTCCAATCCAC GAACTCGATCAGGGCCTCCTCA-2 2 CTGCAACAACACCACCATCCAC GGGGCAGAGGGACTAACCGAC A-3 3AGCTCCTTCACCACTCTCGTG GGACCCTCAGGCGCTGCAAG A-4 3 AGCTCCTTCACCACTCTCGTGGTCAGGAAAGGAGCGGAGCCCATG A-5 4 CCTCTGACTCTAGTCTCTGTGTCGGAGCCAGGCAGGACTGACTC A-6 5 GACCCTACTCTCTCTTTTCCTG CGCCATGGAGCAAGCAGGGAGA-7 6 GCCAACTCTGCTCTCTCTGCAC CCTTCCAGGCCTCCCAGTCAG A-8 7CACGGAATCAGGTTGGGCCTC CACGGAATCAGGTTGGGCCTC A-9 8 CCTCTCCACCCTCCTCCCTTCGGGGCTCCCTGGAGTCTCAC A-10 9 ACAGGCATCTCTCTGTACCCACTGGCTCGGTAACCTGTATTCTAC A-11 10 AACACTGAGCACCTTTCTCCATCACCCATCCCTTCCCCACACTC A-12 11 GACCTTGATGACACCCCCATTCCAGGGACCAGGGCAGGACTG A-13 12 TCTTCCCACCCTCTGTCCCAC CAGGCTCCATCCAGGCACGACA-14 13 AGAACCCTCTGTCCCATCGTC CTGGAGACCAGTATCGGCTTC A-15 13GTCTGTGGTGGAGATGGCTGAG GCCTGGGTGGGACAAGGACAG A-16 13GGTAGCCTCCACCCTGGCATC GCCTTGGTGTGAGAAACCTCTC B-1 1 ATGCCTCTCTGCAGGTGCCACAGCTGTGCACTCCGGGGCCAC B-2 2 TTCCCCCTAACCAGCCCTCTC CATTGCTTGATATGTGCCCAGB-3 3 TGGCCTCCACAGTGTAGCCTC CATCTCTACTAGCTCCTGCTG B-4 3TGGCCTCCACAGTGTAGCCTC CCGACTGTCCGTAGGTGCCAG B-5 4 CCTGCCCTGCAGCTGATGCTGGGTTAAAGCCTCATGGCTCTG B-6 5 CGCAGCCCTCACCCCACCCTC GCCCTTGGGCTCCGGCCATACB-7 6 AAGCAACCCCTCTAAACACAG AGGCGTGCACCACCTTCCCAC B-8 7CCTGTGTTCTCTCATTATGAAC GATCCCCCGTGCCCCCGCTC B-9 8 AACCTCTTGGCCGCCTTTCTGTGTGCCCGCCCACCCGCACTC B-10 9 GCAGGGACCACAACAGGCCTG GTGGTTGCAAAAGTTGCCATCB-11 10 GATGGCAACTTTTGCAACCAC CCAGCCCCGCCCAGGCTCAG B-12 11GGCCCATCTCGCTGCCTCCTG AGGGCTGGGGTATTGGGAGAC B-13 12 CAAGAATGTGTGGCCTGAGAAAGTTGGTGTGGGCCTCCAC B-14 12 CACCAACTTTGGCTTCCAGCC GGCTGCTCAGTGAGTTTCAGB-15 12 CTGGTGGCCTTGGCCAAGAG GTCCAGCGTCTGCAGACGCAG G-1 1GTCCCATCCTCGCCATG CTGCAACATCAACCCCTACAA G-2 2 CCCTCTCCCTGACTTTTCCTCAATGAGAAGGTGAAATCTTACC G-3 3 CGCATCTCCTCTTATTCACAG AGAGCAGCATTCTCTCCTGACG-4 4 GACCCATTTTCTTCCTCCATAG CCTTGGCACAGGTTTCCTTAC G-5 5CAGGTGGTCTTATCCTCCCAG CTCCAAGCCTATGGAAATGAG G-6 6 GAGGACAGGGCTGAGTGTGTGCAGGGCTGGGTGCCCCTGCCA G-7 7 TCCTGGGTCTCCTCTTTCAGA CTGGAGCTGGGTCTCACTCACG-8 8 GCCCTCTCCCTTGTCCCTCAG GTTCCCCACTCTGCCCACCG G-9 9CGCTTTCTCTCTCCGTTGTAG GAACAGGGTAGAGGTAACTTAC G-10 10TTCACCTGTTGGAATTTTGCAG GAAGGAAGCCACTCTACTCAC G-11 11TTGATGGTGTGGCTTGGCCTG TACGGGGAGCTTCTGGACATG G-12 11GCAGAAAGCCAAGGAGTGGTG GATCTGTCTTCTCAACCCTGC Primers are all presented5′-3′. A, B, and G refer to primers for αENaC, βENaC and γENaC,respectively. Primers A13R, A14F, B13R, B14F and R, B15F, G1F and R,G11R and G12F are in coding regions; the remainder are in introns oruntranslated regions. Forward primers A-1 through A-16 correspond to SEQID NO: 21 through SEQ ID NO: 36, respectively. Reverse primers A-1through A-16 correspond to SEQ ID NO: 37 through SEQ ID NO: 52,respectively. Forward primers B-1 through B-15 # correspond to SEQ IDNO: 53 through SEQ ID NO: 67, respectively. Reverse primers B-1 throughB-15 correspond to SEQ ID NO: 68 through SEQ ID NO: 82, respectively.Forward primers G-1 through G-12 correspond to SEQ ID NO: 83 through SEQID NO: 94, respectively. Reverse primers G-1 through G-12 correspond toSEQ ID NO: 95 through SEQ ID NO: 106, respectively.

Discussion

The finding of independent mutations in ENaC subunits that cosegregatewith PHA 1, are homozygous by descent in affected offspring ofconsanguineous union, and result in diminished ENaC activity constituteproof that mutations in subunits of the epithelial sodium channel causeautosomal recessive PHA1.

Thus far, functional mutations in 5 of 7 consanguineous kindreds studiedhave been identified; these mutations occur in either the α or βsubunits of ENaC, demonstrating genetic heterogeneity of the trait. Inthe two kindreds in which mutations have not thus far been identified,one case is homozygous for all markers tightly linked to the β-γ ENaClocus, raising the possibility of an undetected mutation; the other caseis not homozygous for any loci linked to β-γ ENaC, and is homozygous foronly one of two loci tested linked to αENaC. This latter subjectpresented at 8 months of age (Table 1), later than typical PHA1subjects, raising the possibility that this patient might have asomewhat different clinical syndrome. These findings leave open thequestion of whether additional loci will prove to contribute to thepathogenesis of recessive forms of PHA 1.

In contrast to the recessive kindreds described here, some PHA1 kindredshave been reported to show autosomal dominant transmission (Hanukoglu,A. J. Clin. Endocrin. & Metab. 73,936-944 (1991); Limal, J. M., et al.Lancet 1:51 (1978); Hanukoglu, A., et al. Lancet 1:1359 (1978)). SinceENaC is a multimeric channel, it is possible that some ENaC mutationscould have dominant negative function, with one defective gene productsufficient to disrupt normal assembly of a large fraction of channels.Further investigation of such kindreds will be required to evaluate thispossibility.

Knowledge that PHA1 can result from loss of function mutations in ENaCprovides the basis for a detailed understanding the pathogenesis of thisdisease. Affected neonates have a primary defect in renal sodiumreabsorption mediated via this channel. The consequence is salt wasting,leading to intravascular volume depletion; this results in a markedincrease in secretion of renin and consequently aldosterone, in aneffort to restore plasma volume. However, because ENaC is defective,renal sodium reabsorption cannot be appropriately increased, resultingin persistent intravascular volume depletion. In addition, sodiumreabsorption via ENaC is indirectly coupled to K⁺ secretion and H⁺secretion in the distal nephron. As a result, the loss of ENaC functionimpairs the ability to secrete K⁺ and H⁺, contributing to hyperkalaemiaand metabolic acidosis; these features are further worsened by poorperfusion of tissues due to hypovolemia. In addition to this renaldefect, parallel defects altering ENaC function in the colon and sweatglands may further augment salt wasting.

One puzzling clinical feature of PHA1 has been the observation that someaffected children “grow out” of the disease, meaning that at older agesthey can stop supplemental dietary salt. It has been proposed that suchpatients usually if not always have autosomal dominant disease(Hanukoglu, A. J. Clin. Endocrin. & Metab. 73,936-944 (1991)).Consistent with this distinction, the subjects reported here all showrecessive transmission and all remain dependent on supplemental dietarysalt. It will consequently be of interest to determine whether kindredsshowing clear-cut dominant transmission or cases with sporadic diseasewho improve with age harbor mutations in ENaC subunits.

It has recently been appreciated that ENaC plays a major role in theremoval of salt and water from the alveolar space in the lung (Strang,L. B. Physiol. Rev. 71:991-1016 (1991)). This finding has beenemphasized by an αENaC knock-out mouse that shows neonatal lethality dueto respiratory failure, apparently from an inability to clear fluid fromthe alveolar space (Hummler et al. Nature Genet. 1996 (In Press)). Itconsequently is of interest that some PHA 1 patients have concurrentrespiratory problems (Hanukoglu, A., et al. J. Pediatr. 125: 752-755(1994)); interestingly, patient PHA K3-1, who has a truncated αENaC, hasa history of recurrent respiratory infections. Nonetheless, thesepatients do not have a clinical picture of acute respiratory distresssyndrome (ARDS), raising the question of whether the αENaC mutationsresult in complete knock-outs of ENaC activity or whether the portion ofαENaC expressed in these patients is sufficient to provide some residualENaC function in vivo, for example by permitting assembly or targetingof other subunits to the apical membrane. Further investigation of thesechannels and the pulmonary manifestations in these patients willconsequently be of interest.

Identification of the molecular basis of this disease provides the meansfor prenatal genetic testing, which may prove to be of clinical benefitin preventing early death from this disease in kindreds known to besegregating this trait. Affected subjects in all 3 native Saudi PHA1kindreds are homozygous for the identical variant, almost certainly bydescent from a remote common ancestor. Since these kindreds are notknown to be related to one another, this finding suggests that thismutation will prove to be a predominant cause of PHA1 in this country.

These findings bring the number of genes in which mutation causesprimary renal salt wasting in humans to 3- the two genes identifiedherein, and mutations in the thiazide-sensitive sodium-chloridecotransporter that cause Gitelman's syndrome (Simon, D. et al NatureGenet. 12:24-30 (1996)), a disorder characterized by primary renaltubular salt wasting in association with hypokalaemic metabolicalkalosis. These findings underscore the primary role of the kidney inregulating intravascular volume and controlling the ionic composition ofthe vascular space.

Finally, it is noteworthy that mutations in ENaC subunits cause twodiseases: loss of function mutations cause salt wasting and PHA1, whilegain of function mutations cause hypertension and Liddle syndrome. Thatextreme variation in ENaC activity either augments or reduces sodiumreabsorption and blood pressure in humans motivates the furtherexamination of these genes and their regulators in the pathogenesis ofhuman blood pressure variation. The variants listed below, which occurin either the α, β, γ subunits of ENaC, have been obtained from patientssuffering from hypertension.

In the α Subunit of ENaC:

Alanine 334 to threonine

Tryptophan 493 to arginine

Cysteine 618 to phenylalanine

In the β Subunit of ENaC:

Serine 82 to cysteine

Alanine 567 to valine

Isoleucine 586 to histidine

Glycine 589 to serine

Threonine 594 to methionine

Alanine 595 to aspartate

Arginine 625 to cysteine

valine 630 to isoleucine

In the γ Subunit of ENaC:

Threonine 259 to asparagine

Serine 373 to asparagine

Valine 443 to leucine

Proline 502 to alanine

Lysine 570 to asparagine

The functional significance of these mutations has been assessed invitro by expression in Xenopus laevis oocytes. The tryptophan 493 toarginine variant in the α subunit causes marked activation of sodiumtransport, suggesting a functional role in vivo.

A skilled artisan can readily practice the inventions disclosed hereinfollowing the methods and Examples provided herein.

106 1 13 DNA Homo sapiens Segment of wild-type alpha ENaC allele 1ccaccatcca cgg 13 2 11 DNA Homo sapiens Segment of mutant alpha ENaCallele 2 ccaccacacg g 11 3 9 DNA Homo sapiens Segment of wild-type alphaENaC allele 3 ctgtcgcga 9 4 9 DNA Homo sapiens Segment of mutant alphaENaC allele 4 ctgtcacga 9 5 9 DNA Homo sapiens Segment of wild-type betaENaC allele 5 cacggcccc 9 6 9 DNA Homo sapiens Segment of mutant betaENaC allele 6 cacagcccc 9 7 11 PRT Homo sapiens Segment of beta ENaCprotein 7 Cys Asp Asn Thr Asn Thr His Gly Pro Lys Arg 1 5 10 8 11 PRTRattus norvegicus Segment of beta ENaC protein 8 Cys Asn Asn Thr Asn ThrHis Gly Pro Lys Arg 1 5 10 9 11 PRT Xenopus laevis Segment of beta ENaCprotein 9 Cys Asp Asn Thr Asn Thr His Gly Pro Lys Arg 1 5 10 10 11 PRTHomo sapiens Segment of human alpha ENaC protein 10 Cys Asn Asn Thr ThrIle His Gly Ala Ile Arg 1 5 10 11 11 PRT Rattus norvegicus Segment ofalpha ENaC protein 11 Cys Asn Asn Thr Thr Ile His Gly Ala Ile Arg 1 5 1012 11 PRT Xenopus laevis Segment of alpha ENaC protein 12 Cys Ser AsnThr Thr Ile His Gly Ala Ile Arg 1 5 10 13 11 PRT Homo sapiens Segment ofgamma ENaC protein 13 Cys Leu Asn Thr Asn Thr His Gly Cys Arg Arg 1 5 1014 11 PRT Rattus norvegicus Segment of gamma ENaC protein 14 Cys Met AsnThr Asn Thr His Gly Cys Arg Arg 1 5 10 15 11 PRT Xenopus laevis Segmentof gamma ENaC protein 15 Cys Leu Asn Thr Asn Thr His Gly Cys Arg Arg 1 510 16 11 PRT Homo sapiens Segment of delta ENaC protein 16 Cys Thr AsnAla Ile Ile His Gly Ala Ile Arg 1 5 10 17 11 PRT Caenorhabditis elegansSegment of mec-10 protein 17 Cys Tyr Lys Thr Ser Ser His Gly Ile Pro Met1 5 10 18 11 PRT Caenorhabditis elegans Segment of Deg-1 protein 18 CysAsp Lys Thr Thr Ala His Gly Ala Lys Arg 1 5 10 19 21 DNA ArtificialSequence Description of Artificial Sequence Site-directed mutagenesisprimer- forward 19 ccaacacaca cagccccaaa c 21 20 23 DNA ArtificialSequence Description of Artificial Sequence Site-directed mutagenesisprimer- reverse 20 ccttgacctt ggagtactgg aag 23 21 22 DNA ArtificialSequence Description of Artificial Sequence A-1 forward PCR primer 21acccttgctc tctccaatcc ac 22 22 22 DNA Artificial Sequence Description ofArtificial Sequence A-2 forward PCR primer 22 ctgcaacaac accaccatcc ac22 23 21 DNA Artificial Sequence Description of Artificial Sequence A-3forward PCR primer 23 agctccttca ccactctcgt g 21 24 21 DNA ArtificialSequence Description of Artificial Sequence A-4 forward PCR primer 24agctccttca ccactctcgt g 21 25 23 DNA Artificial Sequence Description ofArtificial Sequence A-5 forward PCR primer 25 cctctgactc tagtctctgt gtc23 26 22 DNA Artificial Sequence Description of Artificial Sequence A-6forward PCR primer 26 gaccctactc tctcttttcc tg 22 27 22 DNA ArtificialSequence Description of Artificial Sequence A-7 forward PCR primer 27gccaactctg ctctctctgc ac 22 28 21 DNA Artificial Sequence Description ofArtificial Sequence A-8 forward PCR primer 28 cacggaatca ggttgggcct c 2129 21 DNA Artificial Sequence Description of Artificial Sequence A-9forward PCR primer 29 cctctccacc ctcctccctt c 21 30 22 DNA ArtificialSequence Description of Artificial Sequence A-10 forward PCR primer 30acaggcatct ctctgtaccc ac 22 31 23 DNA Artificial Sequence Description ofArtificial Sequence A-11 forward PCR primer 31 aacactgagc acctttctcc atc23 32 22 DNA Artificial Sequence Description of Artificial Sequence A-12forward PCR primer 32 gaccttgatg acacccccat tc 22 33 21 DNA ArtificialSequence Description of Artificial Sequence A-13 forward PCR primer 33tcttcccacc ctctgtccca c 21 34 21 DNA Artificial Sequence Description ofArtificial Sequence A-14 forward PCR primer 34 agaaccctct gtcccatcgt c21 35 22 DNA Artificial Sequence Description of Artificial Sequence A-15forward PCR primer 35 gtctgtggtg gagatggctg ag 22 36 21 DNA ArtificialSequence Description of Artificial Sequence A-16 forward PCR primer 36ggtagcctcc accctggcat c 21 37 21 DNA Artificial Sequence Description ofArtificial Sequence A-1 reverse PCR primer 37 gaactcgatc agggcctcct c 2138 21 DNA Artificial Sequence Description of Artificial Sequence A-2reverse PCR primer 38 ggggcagagg gactaaccga c 21 39 20 DNA ArtificialSequence Description of Artificial Sequence A-3 reverse primer 39ggaccctcag gcgctgcaag 20 40 24 DNA Artificial Sequence Description ofArtificial Sequence A-4 reverse PCR primer 40 gtcaggaaag gagcggagcc catg24 41 21 DNA Artificial Sequence Description of Artificial Sequence A-5reverse PCR primer 41 ggagccaggc aggactgact c 21 42 21 DNA ArtificialSequence Description of Artificial Sequence A-6 reverse PCR primer 42cgccatggag caagcaggga g 21 43 21 DNA Artificial Sequence Description ofArtificial Sequence A-7 reverse PCR primer 43 ccttccaggc ctcccagtca g 2144 21 DNA Artificial Sequence Description of Artificial Sequence A-8reverse PCR primer 44 cacggaatca ggttgggcct c 21 45 20 DNA ArtificialSequence Description of Artificial Sequence A-9 reverse PCR primer 45ggggctccct ggagtctcac 20 46 23 DNA Artificial Sequence Description ofArtificial Sequence A-10 reverse PCR primer 46 tggctcggta acctgtattc tac23 47 21 DNA Artificial Sequence Description of Artificial Sequence A-11reverse PCR primer 47 acccatccct tccccacact c 21 48 20 DNA ArtificialSequence Description of Artificial Sequence A-12 reverse PCR primer 48cagggaccag ggcaggactg 20 49 21 DNA Artificial Sequence Description ofArtificial Sequence A-13 reverse PCR primer 49 caggctccat ccaggcacga c21 50 21 DNA Artificial Sequence Description of Artificial Sequence A-14reverse primer 50 ctggagacca gtatcggctt c 21 51 21 DNA ArtificialSequence Description of Artificial Sequence A-15 reverse PCR primer 51gcctgggtgg gacaaggaca g 21 52 22 DNA Artificial Sequence Description ofArtificial Sequence A-16 reverse PCR primer 52 gccttggtgt gagaaacctc tc22 53 21 DNA Artificial Sequence Description of Artificial Sequence B-1forward PCR primer 53 atgcctctct gcaggtgcca c 21 54 21 DNA ArtificialSequence Description of Artificial Sequence B-2 forward PCR primer 54ttccccctaa ccagccctct c 21 55 21 DNA Artificial Sequence Description ofArtificial Sequence B-3 forward PCR primer 55 tggcctccac agtgtagcct c 2156 21 DNA Artificial Sequence Description of Artificial Sequence B-4forward PCR primer 56 tggcctccac agtgtagcct c 21 57 21 DNA ArtificialSequence Description of Artificial Sequence B-5 forward PCR primer 57cctgccctgc agctgatgct g 21 58 21 DNA Artificial Sequence Description ofArtificial Sequence B-6 forward PCR primer 58 cgcagccctc accccaccct c 2159 21 DNA Artificial Sequence Description of Artificial Sequence B-7forward PCR primer 59 aagcaacccc tctaaacaca g 21 60 22 DNA ArtificialSequence Description of Artificial Sequence B-8 forward PCR primer 60cctgtgttct ctcattatga ac 22 61 21 DNA Artificial Sequence Description ofArtificial Sequence B-9 forward PCR primer 61 aacctcttgg ccgcctttct g 2162 21 DNA Artificial Sequence Description of Artificial Sequence B-10forward PCR primer 62 gcagggacca caacaggcct g 21 63 21 DNA ArtificialSequence Description of Artificial Sequence B-11 forward PCR primer 63gatggcaact tttgcaacca c 21 64 21 DNA Artificial Sequence Description ofArtificial Sequence B-12 forward PCR primer 64 ggcccatctc gctgcctcct g21 65 19 DNA Artificial Sequence Description of Artificial Sequence B-13forward PCR primer 65 caagaatgtg tggcctgag 19 66 21 DNA ArtificialSequence Description of Artificial Sequence B-14 forward PCR primer 66caccaacttt ggcttccagc c 21 67 20 DNA Artificial Sequence Description ofArtificial Sequence B-15 forward PCR primer 67 ctggtggcct tggccaagag 2068 21 DNA Artificial Sequence Description of Artificial Sequence B-1reverse primer 68 agctgtgcac tccggggcca c 21 69 21 DNA ArtificialSequence Description of Artificial Sequence B-2 reverse PCR primer 69cattgcttga tatgtgccca g 21 70 21 DNA Artificial Sequence Description ofArtificial Sequence B-3 reverse PCR primer 70 catctctact agctcctgct g 2171 21 DNA Artificial Sequence Description of Artificial Sequence B-4reverse PCR primer 71 ccgactgtcc gtaggtgcca g 21 72 21 DNA ArtificialSequence Description of Artificial Sequence B-5 reverse PCR primer 72ggttaaagcc tcatggctct g 21 73 21 DNA Artificial Sequence Description ofArtificial Sequence B-6 reverse PCR primer 73 gcccttgggc tccggccata c 2174 21 DNA Artificial Sequence Description of Artificial Sequence B-7reverse PCR primer 74 aggcgtgcac caccttccca c 21 75 20 DNA ArtificialSequence Description of Artificial Sequence B-8 reverse PCR primer 75gatcccccgt gcccccgctc 20 76 21 DNA Artificial Sequence Description ofArtificial Sequence B-9 reverse PCR primer 76 tgtgcccgcc cacccgcact c 2177 21 DNA Artificial Sequence Description of Artificial Sequence B-10reverse PCR primer 77 gtggttgcaa aagttgccat c 21 78 20 DNA ArtificialSequence Description of Artificial Sequence B-11 reverse PCR primer 78ccagccccgc ccaggctcag 20 79 21 DNA Artificial Sequence Description ofArtificial Sequence B-12 reverse PCR primer 79 agggctgggg tattgggaga c21 80 21 DNA Artificial Sequence Description of Artificial Sequence B-13reverse PCR primer 80 aaagttggtg tgggcctcca c 21 81 20 DNA ArtificialSequence Description of Artificial Sequence B-14 reverse PCR primer 81ggctgctcag tgagtttcag 20 82 21 DNA Artificial Sequence Description ofArtificial Sequence B-15 reverse PCR primer 82 gtccagcgtc tgcagacgca g21 83 17 DNA Artificial Sequence Description of Artificial Sequence G-1forward PCR primer 83 gtcccatcct cgccatg 17 84 21 DNA ArtificialSequence Description of Artificial Sequence G-2 forward PCR primer 84ccctctccct gacttttcct c 21 85 21 DNA Artificial Sequence Description ofArtificial Sequence G-3 forward PCR primer 85 cgcatctcct cttattcaca g 2186 22 DNA Artificial Sequence Description of Artificial Sequence G-4forward PCR primer 86 gacccatttt cttcctccat ag 22 87 21 DNA ArtificialSequence Description of Artificial Sequence G-5 forward PCR primer 87caggtggtct tatcctccca g 21 88 21 DNA Artificial Sequence Description ofArtificial Sequence G-6 forward PCR primer 88 gaggacaggg ctgagtgtgt g 2189 21 DNA Artificial Sequence Description of Artificial Sequence G-7forward PCR primer 89 tcctgggtct cctctttcag a 21 90 21 DNA ArtificialSequence Description of Artificial Sequence G-8 forward PCR primer 90gccctctccc ttgtccctca g 21 91 21 DNA Artificial Sequence Description ofArtificial Sequence G-9 forward PCR primer 91 cgctttctct ctccgttgta g 2192 22 DNA Artificial Sequence Description of Artificial Sequence G-10forward PCR primer 92 ttcacctgtt ggaattttgc ag 22 93 21 DNA ArtificialSequence Description of Artificial Sequence G-11 forward PCR primer 93ttgatggtgt ggcttggcct g 21 94 21 DNA Artificial Sequence Description ofArtificial Sequence G-12 forward PCR primer 94 gcagaaagcc aaggagtggt g21 95 21 DNA Artificial Sequence Description of Artificial Sequence G-1reverse PCR primer 95 ctgcaacatc aacccctaca a 21 96 22 DNA ArtificialSequence Description of Artificial Sequence G-2 reverse PCR primer 96aatgagaagg tgaaatctta cc 22 97 21 DNA Artificial Sequence Description ofArtificial Sequence G-3 reverse PCR primer 97 agagcagcat tctctcctga c 2198 21 DNA Artificial Sequence Description of Artificial Sequence G-4reverse PCR primer 98 ccttggcaca ggtttcctta c 21 99 21 DNA ArtificialSequence Description of Artificial Sequence G-5 reverse PCR primer 99ctccaagcct atggaaatga g 21 100 21 DNA Artificial Sequence Description ofArtificial Sequence G-6 reverse PCR primer 100 cagggctggg tgcccctgcc a21 101 21 DNA Artificial Sequence Description of Artificial Sequence G-7reverse PCR primer 101 ctggagctgg gtctcactca c 21 102 20 DNA ArtificialSequence Description of Artificial Sequence G-8 reverse PCR primer 102gttccccact ctgcccaccg 20 103 22 DNA Artificial Sequence Description ofArtificial Sequence G-9 reverse PCR primer 103 gaacagggta gaggtaactt ac22 104 21 DNA Artificial Sequence Description of Artificial SequenceG-10 reverse PCR primer 104 gaaggaagcc actctactca c 21 105 21 DNAArtificial Sequence Description of Artificial Sequence G-11 reverse PCRprimer 105 tacggggagc ttctggacat g 21 106 21 DNA Artificial SequenceDescription of Artificial Sequence G-12 reverse PCR primer 106gatctgtctt ctcaaccctg c 21

What is claimed is:
 1. A method for identifying a carrier ofpsuedohypoaldosteronism type-1 (PHA-1), comprising the steps of: a)isolating a nucleic acid sample from an individual; and b) analyzing thenucleic acid sample for the presence or absence of a nucleic acidcomprising the nucleotide sequence of SEQ ID NO: 2, 4, or 6, whereby thepresence of any one of the nucleotide sequences is indicative of a PHA-1carrier.
 2. The method of claim 1, further comprising amplification ofthe isolated nucleic acid of step (a), wherein the amplification productis analyzed in step (b).
 3. The method of claim 2, wherein theamplification in step (a) comprises use of a polymerase chain reaction(PCR).
 4. The method of claim 1, wherein the isolated nucleic acid isanalyzed by nucleotide sequencing, selective nucleic acid hybridization,oligonucleotide ligation, RNase mismatch cleavage, restriction fragmentpolymorphism, allele-specific PCR, single strand conformationpolymorphism, electrophoresis, or combinations thereof.
 5. The method ofclaim 2, wherein the amplification product is analyzed by nucleotidesequencing, selective nucleic acid hybridization, oligonucleotideligation, RNase mismatch cleavage, restriction fragment polymorphism,allele-specific PCR, single strand conformation polymorphism,electrophoresis, or combinations thereof.
 6. A method for identifying acarrier of pseudohypoaldosteronism type-1 (PHA-1), comprising the stepsof: a) isolating a nucleic acid sample from an individual; and b)analyzing the nucleic acid sample for the presence or absence of ahomozygous alteration in the nucleotide sequence of SEQ ID NO:2, 4, or6, whereby the presence of the homozygous alteration is indicative of aPHA-1 carrier.